| Objective: Canine infectious hepatitis is an acute and highly contagious viral disease of dogs and puppies, and is caused by canine adenovirus type 1 (CAV-1). This disease is widely distributed in various parts of the world. Dogs, regardless of species, age and gender, can be infected throughout the year. CAV-1 has caused acute-infection and high dying in all breeds of young dogs, which severely harmed the canine cultivation.Currently, vaccinations with attenuated vaccine and modified live virus vaccine provide an effective approach in preventing the disease. However, in the process of vaccine preparation, escaped and incompletely inactivated virus can cause disease outbreaks. In some emergent situation, we can use the homeotype and heterotype of immune serum and the gamma globulin to prevent and treat this disease.Nucleic acid vaccine is a new vaccine technique developed in the field of genetic therapy in 1990s. Nucleic acid vaccines use expression vectors encoding one or more antigen genes to transfect host cells inducing both humoral and cellular immunity against the expressed antigen. Nucleic acid vaccines now has been used to protective antibody and cell-mediated immune responses in a wide variety of preclinical animal models for viral, bacterial, and parasitic diseases. Its advantages such as multivalency, high efficiency, low cost and so on make its potential application value inestimable. So, nucleic acid vaccine may play an epoch-making role in human diseases as well as the healthy development of animal husbandry.We found that vaccination of BALB/c mice with the DNA vaccine pVAX1-CpG-Loop alone resulted in the following consequences: High-level specific antibody (IgG) against ICHV was induced; T cell activation was elicited; and neutralizing antibodies were detectable in immunized mice.To further verify the immune effect of the pVAX1-CpG-Loop in dogs and to find the best dose and immunization strategy. In this study, we immunized Beagles using constructed DNA vaccine of canine infectious hepatitis, and determined its primary immune efficacy. All dogs were challenged on week 6 after last immunization with a hepatitis infection canines virus, and then estimate its immune effect and immunologic memory. We hope our study can explore the possibility of DNA vaccine acting as canine infectious hepatitis vaccine, lay some foundations for deep understanding the immunology of our constructed vaccine.Methods:1 Extracted eukaryotic recombinant plasmid abundantly E.coli that contained the eukaryotic recombinant plasmid pVAX1-CpG-Loop was cultivated for 36h by shake culture method. Then the sediment of E.coli was collected by centrifugalization and recombinant plasmids were extracted by SDS- alkaline lysis. The purity and concentration of the extracted recombinant plasmid were determined by grating spectrophotometer.2 Extraction of recombinant protein antigen LoopWe transformed the eukaryotic expression plasmid pET28a(+) Loop into E.coli BL21(DE3),shaked bacteria and induced the expression of recombination Loop protein. The recombination protein was purified by Ni-NTA affinity chromatography column and condensed after dislysis and renaturation.3 Immunization of dogsSix dogs were randomly numbered 1-6. No.1, 2, 3 dogs were injected with recombinant plasmid pVAX1-CpG-Loop which was diluted with 1ml phosphate buffered saline(PBS, 0.01M, pH 7.2) at doses of 400,600, 800μg respectively and the same recombinant protein antigens Loop on the last time. No.4, 5, 6 dogs were injected with recombinant plasmid pVAX1-CpG-Loop which was diluted with 1ml phosphate buffered saline(PBS, 0.01M, pH 7.2) at doses of 400,600, 800μg respectively. No.7 dog had been vaccinated with modified live virus vaccine.All injections were administered i.m. at the quadriceps muscle. 25% degerming cane sugar 500μl in total was injected into the quadriceps muscle of each dog 15min before immunization. These dogs were immunized after 0,8,16 weeks and challenged by virus 6 weeks after the last immunization.4 Assessment of immune efficacy4.1 T Lymphocyte proliferation-specific analysis MTT were used to evaluate the activity of lymphocyte proliferation.4.2 Cytotoxic T-lymphocyte assay CTL assays were performed using the Cytotox 96 non-radioactive cytotoxicity assay kit.4.3 Lymphocyte-induced cytokines test Induction of cytokines peripheral blood lymphocyte suspensions from immunized dogs were diluted in 10% bovine calf serum-supplemented RPMI 1640 to a final concentration of 5×106 cells/ml. The contents of IFN-γand il-4 in supernatants were detected by ELISA kit.4.4 ELISpot analysis of antigen-activated T cells The response of antigen-specific T cells from immunized dogs was measured by the use of canine IFN-γELISpot sets according to the manufacturer's protocol.4.5 A reverse transcription-polymerase chain reaction technique to detect canine cytokine genes Detecting the expression of IFN-γand IL-4 by RT-PCR.Results:1 The purity and concentration of the recombinant plasmid which extracted abundantly by alkaline lysis detected by grating spectrophotometer dementrated that the ratio of OD260/OD280 was between 1.8 and 2.0. The recombinant Loop proteins have the highest concentration in the GuNTA-200 buffer after purification through Ni-NTA affinity chromatography column.2 At two weeks after the last immunization,the peripheral blood lymphocytes of every immunized dog which was stimulated in vitro by ConA and antigen illustrated higher proliferative activity than that of control dog. Two weeks after virus challenged, after stimulation by ConA, the peripheral blood lymphocytes of immune dog which were immunized by only 800μg plasmid showed higher proliferative activity than the others.3 Protein-specific CTL activity assay results show that at two weeks after the last immunization the LDH release rate of immune dogs which were immunized by plasmid were higher than that of control dog. Two weeks after virus attack, the LDH release rate of immune dogs which were immunized by plasmid plus protein 600μg(pp600) and plasmid 400μg(p400) in the last immunization was higher than that of control dog.4 At the end of two weeks after the last immunization, the peripheral blood lymphocytes of immune dogs were stimulated in vitro by ConA and Loop antigen recombinant protein. Then it could be noticed that the concentration of cytokine'IL-4'in supernatant which from the immune dogs were immunized by plasmid was higher than that of control dog. Two weeks after virus challenged, the concentration of IL-4 in immune dogs which were immunized by only plasmid was a little bit higher than that of control dog. At the end of six weeks after the last immunization, the concentration of cytokine IFN-γin supernatant from immune dogs which were immunized by plasmid was higher than that of control dog. Two weeks after virus challenge, the concentration of cytokine IFN-γin supernatant from immune dogs which were immunized by only plasmid was higher than that of control dog.5 The effector T lymphocytes which excreted IFN-γin the peripheral blood lymphocytes of immune dogs which were immunized by only plasmid 400μg and plasmid plus protein 400μg were more than that of control dog.6 The detection result of the expression of cytokines by RT-PCR indicated that at the end of two weeks after the last immunization and two weeks after virus challenge, the cytokine IL-4 expression of immune dogs which were immunized by plasmid was higher than that of control dog. The expression was significantly increasing after challenge. At the end of two weeks after the last immunization, the cytokine IFN-γwas expressed in immune dogs but not in control dog. After challenge, the cytokine IFN-γwas expressed higher.Conclusion:1 The experimental results show that the proliferative activity of peripheral blood lymphocytes and CTL killing activity can be detected on the dogs immunized by the recombinant plasmid, while the effector T cells which can excrete IFN-γalso appear on these dogs, which illustrates that the recombinant plasmid pVAX1-CpG-Loop can induce the specific cellular immune response after immunizing dogs.2 The concentration of the Cytokine IL-4 in the culture supernatant of the peripheral blood lymphocytes of immune dogs by the recombinant plasmid and its relative expression amount in the mRNA level both come up to the level of control dogs, which illustrates that the recombinant plasmid pVAX1-CpG-Loop can induce humoral immune response after immunizing dogs.3 No obvious discrepancy is noticed between co-immunization with plasmid plus protein and immunization with only plasmid. A good immune effect was obtained through vaccinating Beagle dogs with only recombinant plasmid pVAX1-CpG-Loop. |
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